Vertebrate smooth muscle myosin heavy chain phosphorylation was recently observed in cultured aortic smooth muscle cells as well as in strips of intact aorta by our laboratory (Kawamoto and Adelstein, J. Biol. Chem. 263:1099-1102, 1988). We are now characterizing heavy chain phosphorylation in vitro using purified aortic smooth muscle myosin and purified kinases. We have identified two kinases that phosphorylate the myosin heavy chain. The stoichiometry of phosphorylation by these kinases has been determined, and the sites phosphorylated by the two kinases have been compared by two-dimensional tryptic peptide mapping of the phosphorylated heavy chains. We are currently interested in determining the exact sites of phosphorylation and the effects of heavy chain phosphorylation on myosin filament assembly and MgATPase activity.